Abstract Tuberculosis (TB) is a global health threat, but can be difficult to diagnose and manage in pediatric patients due to the weak performance and non-quantitative nature of frontline diagnostic assays, which function even worse when used with patients co-infected with human immunodeficiency virus (HIV). There is an unmet need for a rapid, non-sputum-based quantitative test to detect active TB cases and anti-TB treatment responses in clinically diverse pediatric populations. The proposed research will overcome these obstacles by developing a solid-state nanopore biosensor assay that can rapidly diagnose pediatric TB by measuring Mycobacterium tuberculosis (Mtb)-secreted antigens in patient serum samples. My solid-state nanopore system is easy to operate, has low fabrication/instrument costs, and can perform high-throughput and ultra-sensitive measurements on specific Mtb-derived peptides. The robust portability of this platform also allows its use in resource-limited areas that are subject to high TB prevalence. We identified two highly Mtb-specific peptide fragments of the Mtb virulence antigens CFP-10 and ESAT-6, and validated their clinical performance as biomarkers for active TB disease diagnosis using a mass spectrometry-based assay with 201 adult and 123 pediatric patients and controls chosen from highly relevant cohorts (e.g. HIV-positive/negative, pulmonary/extrapulmonary, Mtb culture-positive/negative, latent TB, and nontuberculous mycobacteria infections). Similar robust overall diagnostic sensitivities and specificities obtained using these biomarkers in adults (88.6% / 93.8%) and children (88.2% / 97.2%) significantly outperformed those reported for other frontline tests. Quantification of serum Mtb antigen concentration was also informative in monitoring the response to anti-TB treatment in HIV- positive adults. My recent results show that nanopores can accurately detect CFP-10 and/or ESAT-6 peptides in a pilot cohort of pediatric TB cases, and indicate that statistical analysis of nanopore results for peptide profiling holds significant diagnostic promise. Based on these findings, I propose that the portable solid-state nanopore biosensor to be analyzed in these studies can improve TB diagnosis in children, particularly in resource-limited areas with high TB prevalence. I will leverage this system and the robustness of Mtb antigen-derived peptide biomarkers to: (1) develop a nanopore-based TB diagnostic assay; (2) validate this assay in a pediatric cohort with and without HIV co- infection; and (3) determine whether Mtb antigens concentrations in serum decrease in response to treatment in a pilot pediatric cohort during anti-TB treatment. Given the success of these proof-of-concept studies, the long- term goal of the proposed research program is to build prototype devices for large-scale on-site clinical validation studies in high TB burden regions, and to extend this biosensing platform to the detection of other disease biomarkers. This research program should hasten the translation of a promising biosensing method into a practical clinic-ready point-of-care tool for disease diagnosis.